Benzothiazole derivatives and a use thereof for the treatment of cancer

ABSTRACT

Provided are a compound represented by Formula I, a pharmaceutically acceptable salt thereof, or a solvate thereof; and a pharmaceutical composition for cancer treatment and a pharmaceutical composition for a radiation sensitizer for cancer treatment, each pharmaceutical composition including the compound of Formula I, the pharmaceutically acceptable salt thereof, or the solvate thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein, in Formula I, 
             —R 1  is a C 1 -C 3  alkoxy, ═O, or —OH, and 
             —R 2  is a 5- or 6-membered heteroaryl including 1 to 2 hetero atoms selected from nitrogen and oxygen, wherein a carbon of the 5- or 6-memberedheteroaryl is optionally substituted with a C 1 -C 3  alkyl a C 1 -C 3  alkoxy, or hydroxy.

BACKGROUND

1. Field

One or more embodiments of the present invention relate to novelbenzothiazole derivative compounds effective for cancer treatment, andmore particularly, to a benzothiazole derivative compound, a use of thebenzothiazole derivative compound for cancer treatment, and a use of thebenzothiazole derivative compound as a radiation sensitizer for cancertreatment.

2. Description of the Related Art

Even with the recent sharp increase in cancer incidence due to rapidindustrial development, global ecosystem changes, and dietary changes,cancer is still an incurable disease due to the yet unidentifiedincidence mechanism of cancer. Anticancer drugs in current use may belargely classified into biological drugs, such as enzymatic drugs orvaccines, pure synthetic drugs, and drugs derived from natural products.Anticancer drugs may exhibit various pharmacological actions dependingon the types of cancer and have various side effects due to toxicity,and thus, may be problematic in cancer treatment. Anticancer drugs mayeffectively suppress the growth of cancer cells, but also have toxicityto normal cells. Due to this, many scholars have done research todevelop a more effective anticancer drug having minimum toxicity tonormal cells.

Lung cancer is the second most prevalent cancer, next to gastric cancer,and has been first in mortality among other cancers since the year 2000,due to a poor prognosis. Lung cancer may be histologically classifiedinto small-cell lung cancer or non-small cell lung cancer. Chemotherapyand radiotherapy are recommended for small-cell lung cancer, and radicallumpectomy is known as the best treatment for non-small cell lungcancer. Lung cancer may have a difficulty in local tumor control, andmay include micro-metastatic cancer cell that are likely undetectable bydiagnostic imaging. Accordingly, local radiotherapy alone on a lung maylead to metastasis to other remote organs, with a 5-year survival rateof less than 10%. Accordingly, a various combinations of radiotherapywith chemotherapy have been used to prevent the recurrence of cancer inremote organs and have been found to be more effective than radiotherapyonly.

Peroxisome proliferator activated receptors (PPARs) which are nuclearreceptors belonging to the steroid-thyroid-retinoid receptor superfamilyare transcription factors of which activities are regulated by variousligands. PPARs are also key factors to regulate sugar and lipidmetabolisms, and are also known to regulate cell division, celldifferentiation, and cell death in various tissues. Activation of PPARis known to exhibit anticancer activity in various cancers.

Thiazolidinediones as a diabetes treatment drug, including troglitazone(TGZ), ciglitazone, rosiglitazone, or pioglitazone, are synthetic PPARγagonists. Reportedly, TGZ is known to have a cytotoxic effect on varioushuman cancers of the colon (Non-patent document 1), the breast(Non-patent document 2), the liver, the lungs, the kidneys, and theprostate.

It has been suggested that the activation of PPAR β/δ ameliorates lungcancer. High-affinity synthetic ligand for PPAR β/δ, such as L165041,was found to suppress cell proliferation in human lung cancer(Non-patent document 3) and to exacerbate lung cancer in a transgenicmouse lacking the gene expression of PPAR β/δ (Non-patent document 4).

The expression of PPAR γ prognosis was found to be reduced in lungcancer patients with a poor prognosis (Non-patent document 8). Theactivation of PPAR γ by an endogenous agonist or a synthetic agonist wasfound to suppress the growth of lung cancer (Non-patent document 5). Thetreatment of non-small cell lung cancer with PPAR γ active materials wasreported to induce apoptosis and differentiation (Non-patent document6). Ciglitazone was reported to suppress tumors derived from A-549 cellsin nude mice (Non-patent document 7). Diabetes patients administeredwith thiazolidinedione known as PPAR γ agonist to treat diabetes werefound to have a remarkably low likelihood of developing lung cancer(Non-patent document 8). The reaction of PPAR-γ ligands was found toprotect the body from lung cancer (Non-patent documents 9 and 10).

PPAR-γ ligands are known to have anticancer functions through dependentor independent pathways on PPAR-γ, and in particular, the latter wasfound to be related with lung cancer (Non-patent document 11).

PRIOR ART DOCUMENTS List of Non-Patent Documents

1. Sarraf P, Mueller E, Jones D, King F J, DeAngelo D J, Partridge J B,Holden S A et al., Differentiation and reversal of malignant changes incolon cancer through PPARgamma. Nat Med. 1998 September; 4(9):1046-52.

2. Elstner E, Müller C, Koshizuka K, Williamson E A, et al., Ligands forperoxisome proliferator-activated receptorgamma and retinoic acidreceptor inhibit growth and induce apoptosis of human breast cancercells in vitro and in BNX mice. Proc Natl Acad Sci USA. 1998 Jul. 21;95(15):8806-11.

3. Fukumoto K, Yano Y, Virgona N, Hagiwara H, et al., Peroxisomeproliferator-activated receptor delta as a molecular target to regulatelung cancer cell growth. FEBS Lett. 2005 Jul. 4; 579(17):3829-36.

4. Müller-Brüsselbach S, Ebrahimsade S, Jäkel J, Eckhardt J, Rapp U R,Peters J M, et al., Growth of transgenic RAF-induced lung adenomas isincreased in mice with a disrupted PPARbeta/delta gene. Int J Oncol.2007 September; 31(3):607-11.

5. Tsubouchi Y, Sano H, Kawahito Y, Mukai S, Yamada R, Kohno M, Inoue K,et al., Inhibition of human lung cancer cell growth by the peroxisomeproliferator-activated receptor-gamma agonists through induction ofapoptosis. Biochem Biophys Res Commun. 2000 Apr. 13; 270(2):400-5.

6. Chang T H, Szabo E. Induction of differentiation and apoptosis byligands of peroxisome proliferator-activated receptor gamma in non-smallcell lung cancer. Cancer Res. 2000 Feb. 15; 60(4):1129-38.

7. Zhang W, Zhang H, Xing L., Influence of ciglitazone on A549 cellsgrowth in vitro and in vivo and mechanism. J Huazhong Univ Sci TechnolMed Sci. 2006; 26(1):36-39.

8. Govindarajan R, Ratnasinghe L, Simmons D L, Siegel E R, Midathada MV, Kim L, Kim P J, et al., Thiazolidinediones and the risk of lung,prostate, and colon cancer in patients with diabetes. J Clin Oncol. 2007Apr. 20; 25(12):1476-81.

9. Girnun G D, Chen L, Silvaggi J, Drapkin R, Chirieac L R, Padera R F,Upadhyay R, et al., Regression of drug-resistant lung cancer by thecombination of rosiglitazone and carboplatin. Clin Cancer Res. 2008 Oct.15; 14(20):6478-86.

10. Shou Wei Han et al., Anticancer actions of PPARγ ligands: Currentstate and future perspectives in human lung cancer, World J Biol Chem2010 Mar. 26; 1(3): 31-40.

11. Jun-Jie Wang, Oi-Tong Mak. Induction of apoptosis by 15d-PGJ2 viaROS formation: An alternative pathway without PPARγ activation innon-small cell lung carcinoma A549 cells. Prostaglandins & other LipidMediators 2011; 94; 104-111.

SUMMARY

One or more embodiments of the present invention include novel compoundsable to function as PPAR-γ ligands and radiation sensitizers andanticancer agents.

One or more embodiments of the present invention include pharmaceuticalcompositions for cancer treatment that include the novel compounds.

One or more embodiments of the present invention include pharmaceuticalcompositions for a radiation sensitizer for cancer treatment thatinclude the novel compounds.

Additional aspects will be set forth in part in the description whichfollows and, in part, will be apparent from the description, or may belearned by practice of the presented embodiments.

According to one or more embodiments of the present invention, there isprovided a compound of Formula I, a pharmaceutically acceptable saltthereof, or a solvate thereof:

wherein, in Formula I,

—R¹ is a C₁-C₃ alkoxy, ═O or —OH, and

—R² is a 5- or 6-membered heteroaryl including 1 to 2 hetero atomsselected from nitrogen and oxygen, wherein a carbon of the 5- or6-membered heteroaryl is optionally substituted with a C₁-C₃ alkyl, aC₁-C₃ alkoxy, or hydroxy.

According to one or more embodiments of the present invention, apharmaceutical composition for cancer treatment includes a compound ofFormula 1, a pharmaceutically acceptable salt thereof, or a solvatethereof according to the above-description.

According to one or more embodiments of the present invention, apharmaceutical composition for a radiation sensitizer for cancertreatment includes a compound of Formula 1, a pharmaceuticallyacceptable salt thereof, or a solvate thereof according to theabove-description.

BRIEF DESCRIPTION OF THE DRAWINGS

These and/or other aspects will become apparent and more readilyappreciated from the following description of the embodiments, taken inconjunction with the accompanying drawings in which:

FIG. 1 illustrates the results of imaging degrees of adipogenesis viaOil-Red-O staining after treatment of 3T3-L1 cells with controlcompounds and 10 μM of compounds PB01 or PB11 of Examples 3 and 5 forabout 48 hours (2 days) to induce adipogenesis;

FIG. 2A to FIG. 2C illustrate the results of measuring cell viability byWST-1 method after incubation of various cancer cell strains with thecompound PB01 of Example 5;

FIG. 2D to FIG. 2F illustrate the results of measuring cell viability byWST-1 method after incubation of various cancer cell strains with thecompound PB11 of Example 3;

FIG. 3A illustrates graphs of cell viability with respect to testcompound concentration and incubation time in non-small cell lung cancercell strains A549 and H460 after incubation together with the compoundsPB01 or PB 11 of Examples 5 and 3, obtained by microscopy with trypanblue staining;

FIG. 3B illustrates graphs of cell viability with respect to testcompound concentration and incubation time in the non-small cell lungcancer cell strain H460 after incubation together with the compoundsPB01, PB 11, or PB12 of Examples 5, 3, and 4, obtained by microscopywith trypan blue staining;

FIG. 4 illustrates microscopic images of the non-small cell lung cancercell strain H460 after incubation with 50 μM of the compounds PB01 orPB11 of Examples 3 and 5 for about 48 hours, obtained using an invertedmicroscope at a magnification of 200×.

FIG. 5 illustrates fluorescent microscopic images of the non-small celllung cancer cell strain H460 at a 400× magnification obtained viaHoechst staining after incubation with 50 uM of the compounds PB01 orPB11 of Examples 5 and 3 for about 48 hours in order to observe nuclearmorphological changes in the cancer cells.

FIG. 6 illustrates graphs of degrees of nuclear agglutination andsegmentation, obtained based on the nuclear morphological changes in thenon-small cell lung cancer cell strain A549 or H460 that were observedby fluorescent microscopy at a 400× magnification via Hoechst stainingafter incubation with 50 uM of the compound PB01 or PB11 of Examples 5and 3 for about 48 hours.

FIG. 7 illustrates graphs of amount of lactate dehydrogenase (LDH)released from the non-small cell lung cancer cell strains A549 and H460treated with 50 uM of the compound PB01 or PB11 of Examples 5 and 3 intoculture media while incubation for 0 to 48 hours;

FIG. 8A illustrates the results of flow cytometry on the non-small celllung cancer cells A549 after incubation with 50 μM of the compounds ofPB01 or PB11 of Examples 5 and 3 for 48 hours, followed by staining withpropidium iodide;

FIG. 8B illustrates the results of flow cytometry on the non-small celllung cancer cells H460 after incubation with 50 μM of the compounds ofPB01 or PB11 of Examples 5 and 3 for 48 hours, followed by staining withpropidium iodide;

FIG. 9A illustrates graphs of apoptosis ratio in the non-small cell lungcancer cell strains A549 and H460 incubated with 50 μM of the compoundsPB01 or PB11 of Examples 5 and 3 for about 40 hours, wherein theapoptosis ratios were calculated based on the results of flow cytometryusing FACScan™ (available Becton Dicknson) via staining with an AnnexinV Flous Staining kit in a dark condition for about 30 minutes;

FIG. 9B illustrates graphs of apoptosis ratio in the non-small cell lungcancer cell strain H460 incubated with the compounds PB01, PB11, or PB12of Examples 5, 3, and 4 for about 40 hours, wherein the apoptosis ratioswere calculated based on the results of flow cytometry using FACScan™(available Becton Dicknson) via staining with an Annexin V FlousStaining kit in a dark condition for about 30 minutes;

FIG. 10 illustrates images obtained from western blotting followingsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) onproteins extracted from the non-small cell lung cancer cell strain H460incubated with 50 μM of the compounds PB01 or PB11 of Examples 5 and 3for about 48 hours.

FIG. 11 is a graph illustrating the results of calculating colonyformation ratio in the non-small cell lung cancer cell strain H460 on aplate after treatment with ciglitazone (10 μM), PB01(10 μM), or PB11 (5μM,10 μM), irradiation with γ-rays, and then incubation for 14 days;

FIG. 12 illustrates data obtained based on the results of colonycounting of the non-small cell lung cancer cell strain H460 on a plateafter treatment with ciglitazone (10 μM), PB01(10 μM), or PB11 (5 μM,10μM), irradiation with γ-rays, and then incubation for 14 days; and

FIG. 13 illustrates visually observed images of colony formation in thenon-small cell lung cancer cell strain H460 on a plate after treatmentwith ciglitazone (10 μM), PB01(10 μM), or PB11 (5 μM, 10 μM),irradiation with γ-rays, and then incubation for 14 days.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings, wherein like referencenumerals refer to like elements throughout. In this regard, the presentembodiments may have different forms and should not be construed asbeing limited to the descriptions set forth herein. Accordingly, theembodiments are merely described below, by referring to the figures, toexplain aspects of the present description. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items. Expressions such as “at least one of,” whenpreceding a list of elements, modify the entire list of elements and donot modify the individual elements of the list.

Unless otherwise defined, all terms, including technical and scientificterms, used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It willalso be appreciated that, although exemplary methods or compounds aredescribed herein, all similarities or equivalents to the exemplaryembodiments herein that do not depart from the spirit and scope of thepresent disclosure are encompassed in the present invention. Thedisclosures of reference documents, including non-patent documents,referred to herein are incorporated herein in their entirety byreference.

As a result of research into the development of novel compoundsfunctioning as PPAR-γ ligands with anticancer activity, the presentinventors developed novel benzothiazole derivative compounds.

According to an aspect of the present disclosure, there are provided acompound represented by Formula 1, a pharmaceutically acceptable saltthereof, or a solvate thereof:

In Formula I,

—R¹ may be a C₁-C₃ alkoxy, ═O or —OH, and

—R² may be a 5- or 6-membered heteroaryl including 1 to 2 hetero atomsselected from nitrogen and oxygen, wherein a carbon of the 5- or6-membered heteroaryl is optionally substituted with a C₁-C₃ alkyl, aC₁-C₃ alkoxy, or hydroxy.

For example, the compound of Formula I may be selected from the groupconsisting of 4-methoxy-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isooxazole-4-yl)sulfanyl-benzothiazole-6-yl]-amide,4-oxo-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isoxazole-4-yl)sulfanyl-benzothiazole-6-yl]-amide, and4-hydroxy-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isoxazole-4-yl)sulfanyl-benzothiazole-6-yl]-amide.

The pharmaceutically acceptable salt may be present as an acid additionsalt with free acid. The compound of Formula I may form apharmaceutically acceptable acid addition salt according to a commonmethod known in the art. The free acid may be organic acid or inorganicacid. For example, the inorganic salt may be hydrochloric acid, bromicacid, sulfuric acid, or phosphoric acid. For example, the organic acidmay be citric acid, acetic acid, lactic acid, tartaric acid, maleicacid, fumaric acid, formic acid, propionic acid, oxalic acid,trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid,glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid,embonic acid, glutamic acid, or aspartic acid

The pharmaceutically acceptable salt may be present as an inorganic saltof the compound of Formula I. The compound of Formula I may form apharmaceutically acceptable inorganic salt according to a common methodknown in the art. The inorganic salt may be a salt of aluminum,ammonium, calcium, copper, iron, lithium, magnesium, manganese,potassium, sodium, or zinc, but is not limited thereto. For example, theinorganic salt may be a salt of ammonium, calcium, magnesium, potassium,or sodium.

The compound of Formula I may be in the form of any salt, hydrate, orsolvate that may be prepared using a common method in the art, inaddition to such a pharmaceutically acceptable salt as described above.

The compound of Formula I may be synthesized by a method as representedin Reaction Scheme I.

In Reaction Scheme I, R³ and R⁴ in Formula V may be each independentlyhydrogen, a C₁-C₃ alkyl, a C₁-C₃ alkoxy, or hydroxy. In Reaction SchemeI, R⁵ may be a C₁-C₃ alkyl.

The synthesis method illustrated in Reaction Scheme I is described inmore detail in Examples 1 to 5 described later. Although Examples 1 to 5are described with specific functional groups for R³, R⁴, and R⁵ ofFormula V, it may be obvious to one of ordinary skill in the art oforganic chemistry to change functional groups for R³, R⁴, and R⁵ basedon Reaction Scheme I and the synthesis method described in the examples.Although some embodiments of preparing the compound of Formula I aredescribed herein, it may be obvious to one of ordinary skill in the artof organic chemistry to prepare the compound of Formula I differentlythan described herein by appropriately changing starting materials,reaction pathways, and reaction conditions.

The compound of Formula I was identified to be a PPAR γ ligand byOil-Red-O staining (Example 6), and was found to suppress the growth ofvarious cancer cells, including human colorectal cancer cells, breastcancer cells, non-small cell lung cancer cells, and leukemia cells, andin particular, to be more sensitive to suppress the growth of anon-small cell lung cancer cell strain, selectively only to cancercells, without growth suppression or killing of non-cancer lung cells(Example 7). When treated with the compound of Formula I, cancer cellslost adhesion, with an increase in apoptotic body by chromaticagglutination and an increase in extracellular lactate dehydrogenase(LDH), indicating that the death of cancer cells is from apoptosis(Example 8). When used along with radiotherapy, the compound of FormulaI was shown to have a dose increase rate of 1 or greater or 2 or less(Example 10), indicating that the compound of Formula I may be effectiveas a radiation sensitizer for cancer treatment.

According to another aspect of the present disclosure, there areprovided a pharmaceutical composition for cancer treatment that includesa compound of Formula 1 below, a pharmaceutically acceptable saltthereof, or a solvate thereof:

In Formula I,

—R¹ may be a C₁-C₃ alkoxy, ═O or —OH, and

—R² may be a 5- or 6-membered heteroaryl including 1 to 2 hetero atomsselected from nitrogen and oxygen, wherein a carbon of the 5- or6-membered heteroaryl is optionally substituted with a C₁-C₃ alkyl, aC₁-C₃ alkoxy, or hydroxy

According to another aspect of the present disclosure, there is provideda pharmaceutical composition for a radiation sensitizer for cancertreatment, the pharmaceutical composition including a compound ofFormula 1, a pharmaceutically acceptable salt thereof, or a solvatethereof as described above.

For example, the cancer may be bladder cancer, gastric cancer,colorectal cancer, esophageal cancer, pancreatic cancer, lung cancer,non-small cell lung cancer, colon cancer, bone cancer, skin cancer, skinor ocular melanoma, uterine cancer, rectal cancer, anal cancer, breastcancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngealcancer, laryngeal cancer, brain cancer, leukocytes cancer, prostatecancer, kidney or ureter cancer, renal cell carcinoma, renal pelviccarcinoma, central nervous system (CNS) tumors, primary CNS lymphoma,spinal cord tumors, brainstem gliomas, or pituitary adenomas, but is notlimited thereto. In some embodiments, the cancer may be lung cancer,breast cancer, colorectal cancer, or leukemia, and in some otherembodiments, non-small cell lung cancer.

The pharmaceutical composition may be prepared in any commonpharmaceutical dosage form known in the art. For example, thepharmaceutical dosage form may include any form of oral preparations,injections, suppositories, preparations for percutaneous administrationor nasal administration, but is not limited thereto. For example, thepharmaceutical composition may be prepared as oral preparations orinjections.

In formulating the pharmaceutical composition in any of the above-listeddosage forms, a pharmaceutically acceptable carrier appropriate for eachdosage form may be further added. As used herein, the term“pharmaceutically acceptable carrier” refers to any additive ingredientsexcluding the pharmaceutically active ingredient. The term“pharmaceutically acceptable” refers to the properties that do not causeany pharmaceutically undesirable change via interaction betweeningredients of the oral pharmaceutical compositions (for example, viainteraction between carriers or via interaction between thepharmaceutically active ingredient and a carrier). Selection of thepharmaceutically acceptable carrier may be dependent on the propertiesand the administration method of a particular dosage form, the effectsof the carrier on solubility and stability of the dosage form, and thelike.

In some embodiments, the pharmaceutically acceptable carrier containedin the pharmaceutical composition for oral administration may be atleast one selected from the group consisting of a diluent, a binder, aglidant (or a lubricant), a disintegrant, a stabilizer, a solubilizingagent, a sweetening agent, a coloring agent, and a flavoring agent.

The diluent refers to any excipient added to increase the volume of theoral pharmaceutical composition to formulate it into a target dosageform with an appropriate size. Non-limiting examples of the diluent maybe starch (for example, potato starch, corn starch, wheat starch,pregelatinized starch), microcrystalline cellulose (for example,low-hydration microcrystalline cellulose), lactose (for example, lactosemonohydrate, anhydrous lactose, spray lactose), glucose, sorbitol,mannitol, sucrose, alginate, alkaline earth metal salts, clay,polyethylene glycol, dicalcium phosphate, anhydrous calciumhydrogenphosphate, or silicon dioxide, which may be used alone or as amixture thereof. In some embodiments, the excipient may be used fromabout 5 wt % to about 50 wt % based on a total weight of thepharmaceutical composition for oral administration. In some otherembodiments, the excipient may be used from about 10 wt % to about 35 wt% based on the total weight of the pharmaceutical composition forappropriate tabletting and quality maintenance.

The binder refers to a material that offers materials in powder formadhesiveness and facilitates compression of the materials. The bindermay be at least one selected from among starch, microcrystallinecellulose, highly dispersible silica, mannitol, lactose, polyethyleneglycol, polyvinylpyrrolidone, cellulose derivatives (for example,hydroxypropyl methylcellulose, hydroxypropyl cellulose, orlow-substituted hydroxypropyl cellulose), natural gum, synthetic gum,povidone, co-povidone, and gelatin, but is not limited thereto. In someembodiments, the binder may be used from about 2 wt % to about 15 wt %based on a total weight of the pharmaceutical composition for oraladministration. In some other embodiments, the binder may be used fromabout 1 wt % to about 3 wt % based on the total weight of the oralpharmaceutical composition for appropriate tabletting and qualitymaintenance.

The disintegrant refers to a material added to facilitate disintegrationor dissolution of a solid dosage form when administrated into a livingbody. The disintegrant may be starch, such as sodium starch glycolate,corn starch, potato starch, or pregelatinized starch, or modifiedstarch; clay, such as bentonite, montmorillonite, or veegum; cellulose,such as microcrystalline cellulose, hydroxypropyl cellulose, orcarboxymethyl cellulose; an algin, such as sodium alginate or alginicacid; a cross-linked cellulose, such as croscarmellose sodium; gum suchas guar gum or xanthan gum; a cross-linked polymer such as cross-linkedpolyvinylpyrrolidone (crospovidone); or an effervescent agent such assodium bicarbonate or citric acid, which may be used alone or as amixture thereof, but is not limited thereto. In some embodiments, thedisintegrant may be used from about 2 wt % to about 15 wt % based on atotal weight of the pharmaceutical composition for oral administration.In some other embodiments, the disintegrant may be used from about 4 wt% to about 10 wt % based on the total weight of the oral pharmaceuticalcomposition for appropriate tabletting and quality maintenance.

The glidant or lubricant refers to a material that prevents cohesion ofpowders to a compressing system and improves flowability of granules.The glidant may be hard anhydrous silicic acid, talc, stearic acid, ametal salt (magnesium salt, calcium salt, or the like) of stearic acid,sodium lauryl sulfate, hydrogenated vegetable oil, sodium benzoate,sodium stearyl fumarate, glyceryl behenate, glyceryl monostearate, orpolyethylene glycol, which may be used alone or as a mixture thereof,but is not limited thereto. In some embodiments, the glidant may be usedfrom about 0.1 wt % to about 5 wt % based on a total weight of the oralpharmaceutical composition. In some other embodiments, the glidant maybe used from about 1 wt % to about 3 wt % based on the total weight ofthe oral pharmaceutical composition for appropriate tabletting andquality maintenance.

The adsorbent may be hydrated silicon dioxide, hard anhydrous silicicacid, colloidal silicon dioxide (Aerosil, available from Degussa),magnesium aluminometasilicate, microcrystalline cellulose, lactose, or across-linked polyvinylpyrrolidone, which may be used alone or as amixture thereof, but is not limited thereto.

The stabilizer may be at least one selected from the group consisting ofantioxidants, such as butylhydroxyanisole, butylhydroxytoluene,carotene, retinol, ascorbic acid, tocopherol, tocopherol polyethyleneglycol succinic acid, or propyl gallate; cyclic sugar compounds such ascyclodextrin, carboxyethyl cyclodextrin, hydroxypropyl cyclodextrin,sulfobutyl ether, or cyclodextrin; and organic acids such as phosphoricacid, lactic acid, acetic acid, citric acid, tartaric acid, succinicacid, maleic acid, fumaric acid, glycolic acid, propionic acid, gluconicacid, or glucuronic acid, but is not limited thereto.

In some other embodiments, an additive known to improve the taste of byboosting a taste sense may be included. In some embodiments, a sweetenersuch as sucralose, sucrose, fructose, erythritol, acesulfame potassium,sugar alcohol, honey, sorbitol, or aspartame may be added to moreeffectively mask bitterness and maintain the stability and quality ofthe formulation. In some other embodiments, an acidifier such as citricacid or sodium citrate; a natural flavoring such as Japanese apricotflavor, lemon flavor, pineapple flavor, or herbal flavor; or a naturalpigment such as natural fruit juice, chlorophyllin, or flavonoid may beused.

The pharmaceutical composition for oral administration may be a solid,semi-solid, or liquid dosage form acceptable for oral administration.Non-limiting examples of the oral solid dosage form are tablets, pills,hard or soft capsules, powders, fine granules, granules, powders forreconstitution of solution or suspension, lozenges, wafers, oral strips,dragees, or chewable gum, but are not limited thereto. Non-limitingexamples of the oral liquid formulation are solution, suspension,emulsion, syrup, elixir, spirit, aromatic water, lemonade, extract, andtincture. Non-limiting examples of the semi-solid form are aerosol,cream, and gel.

In some embodiments, the pharmaceutical composition may be prepared asinjections. In preparing the pharmaceutical composition as injections, anontoxic buffer solution that is isotonic with blood may be furtheradded as a diluent. An example of the nontoxic buffer solution is aphosphoric acid buffer solution at pH 7.4. The pharmaceuticalcomposition may further include any other diluents or additives, inaddition to the buffer solution.

A carrier for each of the above-listed dosage forms of thepharmaceutical composition and a method of preparing the same may beselected from those widely known in the art, for example, may beprepared according to a method described in the book entitled“Remington's Pharmaceutical Sciences (Newest edition)”.

In some embodiments, a dose and administration time of thepharmaceutical composition for cancer treatment according to any of theabove-described embodiments may vary depending on the age, gender, andbody weight of a target subject, administration route, administrationfrequency, and form of medicine. A total daily dose of thepharmaceutical composition may be about 1 mg/kg to about 1000 mg/kg, andin some embodiments, about 0.01 mg/kg to about 100 mg/kg. The totaldaily dose of the pharmaceutical composition may be appropriately varieddepending to the type of cancer, degree of cancer progression,administration route, and gender, age, and body weight of the targetsubject.

In some embodiments, to enhance radiotherapy effects in cancertreatment, the pharmaceutical composition for a radiation sensitizer forcancer treatment may be administered several times a day in a totaldaily dose of about 1 mg/kg to about 1000 mg/kg for adults as aneffective component. The total daily dose of the pharmaceuticalcomposition for a radiation sensitizer may be appropriately varieddepending to the type of cancer, degree of cancer progression,administration route, and gender, age, body weight, health, or the likeof the target subject.

In any of the pharmaceutical compositions for cancer treatment and thepharmaceutical compositions for a radiation sensitizer, according to theabove-described embodiments of the present disclosure, the amount of thecompound of Formula I may be in a range of about 0.0001 wt % to about 10wt %, and in some embodiments, about 0.001 wt % to about 1 wt %, basedon a total weight of the pharmaceutical composition.

One or more embodiments of the present invention will now be describedin detail with reference to the following examples. However, theseexamples are only for illustrative purposes and are not intended tolimit the scope of the one or more embodiments of the present invention.

EXAMPLE 1 Preparation of Compound of Formula III

2.5 g (43.5 mmol) of a sodium hydroxide aqueous solution was added to asolution of 5.0 g (29.0 mmol) of ethyl-4-cyclohexanone carboxylate inmethanol. The resulting reaction mixture was stirred at room temperaturefor reaction. After completion of the reaction, the solvent was removedunder vacuum to obtain a crude product, which was then dissolved inwater, and adjusted to a pH of 3 to 4 with hydrochloric acid, followedby extraction with ethyl acetate, drying with sodium sulfate (Na₂SO₄),and then concentration. The resulting crude product was purified usingsilica gel column chromatography to obtain 3.5 g of 4-cyclohexanonecarboxylic acid in white solid as a compound of Formula III (Yield:85%).

¹H NMR (CDCl₃, 400 MHz): δ 10.50 (1H, s), 2.62-2.72 (1H, m), 2.30-2.10(4H, m), 2.01-1.86 (4H, m); ¹³C NMR (CDCl₃, 100 MHz): δ 210.8, 180.0,44.5, 40.4(2C), 25.9(2C).

EXAMPLE 2 Preparation of Compound of Formula VI

1.0 g (5.49 mmol) of 6-amino-2-mercapto benzothiazole (IV), 0.749 mL(6.04 mmol) of 4-(chloromethyl)-3,5-dimethylisooxazole (V), and 1.8 g(13.72 mmol) of K₂CO₃ were refluxed in acetone for about 8 hours. Aftercompletion of the reaction, the solvent was removed by rotaryevaporation to obtain a crude product, which was then dissolved inwater, followed by extraction with ethyl acetate, washing with sodiumhydrogen carbonate, drying with sodium sulfate (Na₂SO₄), and thenconcentration. The resulting crude product was purified using silica gelcolumn chromatography to obtain 1.07 g of a compound of Formula VI inwhite solid (Yield: 67%).

¹H NMR (CDCl₃, 400 MHz): δ 7.62 (1H, d, J=8.8 Hz), 6.93 (1H, d, J=2.0Hz), 6.74 (1H, dd, J=2.4, 8.4 Hz), 4.21 (2H, s), 3.87 (2H, s), 2.34 (3H,s), 2.27 (3H, s); ¹³C NMR (CDCl₃, 100 MHz): δ 164.2, 159.9, 158.9,146.6, 143.5, 135.9, 122.6, 119.4, 114.1, 105.4, 100.5, 19.4, 11.0, 6.3.

EXAMPLE 3 Preparation of Compound PB11 of Formula Ia

3.12 g (21.9 mmol) of 4-cyclohexanone carboxylic acid (III), 9.169 g(24.1 mmol) of HBTU, and 7.65 mL (43.8 mmol) ofN,N-diisopropylethylamine (DIEA) were added to a solution of 6.4 g (21.9mmol) of the amine compound of Formula VI in dimethylformate (DMF). Theresulting reaction mixture was stirred at room temperature overnight.After removing DMF under vacuum, the residue was diluted in ethylacetate, washed with sodium carbonate and then brine, dried usinganhydrous sodium sulfate, and then filtered. After removing the solventunder vacuum, the resulting crude product was purified using silica gelcolumn chromatography to obtain 7.0 g 4-oxo-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isoxazole-4-yl)sulfanyl-benzothiazole-6-yl]-amide inwhite solid as a compound of Formula Ia (Yield: 77%).

¹H NMR (DMSO, 400 MHz): δ 10.23 (1H, s), 8.41 (1H, d, J=2.0 Hz), 7.80(1H, d, J=8.8 Hz), 7.55 (1H, dd, J=2.0, 9.2 Hz), 4.38 (2H, s), 2.87-2.79(1H, m), 2.49-2.10 (6H, m), 2.41 (3H, s), 2.24 (3H, s), 1.90 (2H, q,J=13.2 Hz); ¹³C NMR (DMSO, 100 MHz) δ 210.8, 173.0, 164.2, 159.9, 158.9,149.1, 136.5, 135.3, 122.0, 119.4, 110.7, 100.5, 42.6, 40.0(2C),26.8(2C), 19.4, 11.0, 6.3.

EXAMPLE 4 Preparation of Compound PB12 of Formula Ib

0.76 g (2.02 mmol) of NaBH₄ was added to a solution of 0.7 g (1.68 mmol)of the ketone derivative of Formula Ia (Example 3) in ethanol. Theresulting reaction mixture was stirred at room temperature. Aftercompletion of the reaction, the reaction solvent was removed undervacuum to obtain a crude product. This crude product was dissolved inwater, and pH-adjusted with 1N HCl to a pH 6 to 7. The resulting crudeproduct was extracted with ethyl acetate, dried using sodium sulfate(Na₂SO₄), and then concentrated. The resulting crude product waspurified using silica gel column chromatography to obtain 0.3 g of4-hydroxy-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isoxazole-4-yl)sulfanyl-benzothiazole-6-yl]-amide inwhite solid as a compound of Formula Ib (Yield: 43%).

¹H NMR(CD₃OD, 400 MHz): δ 8.25 (1H, d, J=2.0 Hz), 7.73 (1H, d, J=9.2Hz), 7.47 (1H, dd, J=2.4, 8.8 Hz), 4.32 (2H, s), 3.96-3.94 (1H, m),2.43-2.41 (1H, m), 2.39 (3H, s), 2.27 (3H, s), 2.06 (2H, q, J=12.8 Hz),1.87-1.83 (2H, m), 1.66-1.56 (4H, m);

¹³C NMR(CD₃OD, 100 MHz) δ 173.0, 164.2, 159.9, 158.9, 149.1, 136.5,135.3, 122.0, 119.4, 110.7, 100.5, 72.3, 43.5, 33.3(2C), 23.1(2C), 19.4,11.0, 6.3.

EXAMPLE 5 Preparation of Compound PB01 of Formula Ic

0.021 g (0.528 mmol) of NaH and 0.029 mL (0.48 mmol) of methyl iodidewere added to a solution of 0.2 g (0.48 mmol) of the hydroxy derivativeFormula Ib (Example 4) in THF. The resulting reaction mixture wasstirred at room temperature. After completion of the reaction, thereaction solvent was removed by rotary evaporation, followed bydissolving the resulting mixture in water. The resulting mixture wasextracted with ethyl acetate, washed with sodium hydrogen carbonate,dried using sodium sulfate (Na₂SO₄), and then concentrated. Theresulting crude product was purified using silica gel columnchromatography to obtain 0.080 g of 4-methoxy-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isooxazole-4-yl)sulfanyl-benzothiazole-6-yl]-amide inwhite solid as a compound of Formula Ic (where R⁵ is methyl) (Yield:40%).

¹H NMR(CDCl₃, 400 MHz): δ 7.88 (1H, d, J=8.4 Hz), 7.58 (1H, d, J=2.4Hz), 7.24 (1H, dd, J=2.4, 8.8 Hz), 4.36 (2H, s), 3.56-3.54 (1H, m), 3.26(3H, s), 2.48 (3H, s), 2.34 (3H, s), 2.11-2.08 (1H, m), 1.92-1.53 (6H,m), 0.96 (2H, q, J=13.2 Hz); ¹³C NMR(CDCl₃, 100 MHz) δ 173.0, 164.2,159.9, 158.9, 149.1, 136.5, 135.3, 122.0, 119.4, 110.7, 100.5, 84.1,57.1, 43.5, 30.8(2C), 23.4(2C), 19.4, 11.0, 6.3.

EXAMPLE 6 PPAR-γ Ligand Identification Test

The ability to induce adipogenesis of the compound PB11 of Example 3 andthe compound PB01 of Example 5 was tested to identify whether thecompounds PB11 and PB01 had activity of PPAR-γ ligands. 3T3-L1 cells(American Type Culture Collection, Manassas, Va.) were treated with 10μM of the compounds PB11 or PB01 to induce adipogenesis. 3T3-L1 cellswithout any treatment, those treated with the same concentration ofciglitazone, and those treated with the same concentration of GW9662(Cayman, Ann Arbor, Mich.) known as PPAR-γ ligand antagonist were usedas control groups. After the induction of adipogenesis for about 48hours, a degree of adipogenesis was identified by Oil-Red-O staining.The results are shown in FIG. 1.

FIG. 1 illustrates the results of imaging degrees of adipogenesis viaOil-Red-O staining after the treatment of 3T3-L1 cells with controlcompounds and 10 μM of the compounds PB01 or PB11 of Examples 3 and 5for about 48 hours to induce adipogenesis.

As a result of the Oil-Red-O staining, the compound PB01 of Example 5was found to have outstanding ability to induce adipogenesis, which isknown as feature of PPAR-γ ligands. The compound PB11 of Example 3 wasalso found to have high activity in adipogenesis induction. Theseresults indicate that the compounds PB01 and PB11 of Examples 5 and 3were found to be PPAR-γ ligands.

EXAMPLE 7 Evaluation of Cancer Cell Growth Inhibitory Effect

Cancer cell growth inhibitory effects of the compounds PB01 and PB11 ofExamples 5 and 3 identified as PPAR-γ ligands were evaluated. Growthinhibitory effects of the compounds PB01 and PB11 of Examples 5 and 3 onvarious cancer cells, including human non-small cell lung cancer cellstrains (A549, H460), breast cancer cell strains (MCF-7, T-470),colorectal cancer cell strains (LoVo, SW480), and leukocytes cancer cellstrains (HL-60, K562) (American Type Culture Collection, Manassas, Va.),were evaluated using WST-1 method. About 5×10³ cells per well of eachtype of cancer cell strains were portioned into a 96-well plate, andincubated with different concentrations of each of the compounds PB01and PB11 of Examples 5 and 3 in 200 ul of a culture solution for 24hours, 48 hours, and 72 hours. After the incubation for the differentdurations, 10 ul of a WST-1 solution was added to each well, followed byincubation at about 37° C. for about 1 hour. Absorbance at 450 nm wasmeasured to determine cell viability. The results are shown in FIGS. 2Ato 2F.

FIGS. 2A to 2C illustrates the results of measuring cell viability byWST-1 method after the incubation of various cancer cell strains withthe compound PB01 of Example 5.

FIGS. 2D to 2F illustrates the results of measuring cell viability byWST-1 method after the incubation of various cancer cell strains withthe compound PB11 of Example 3.

The growth inhibitory effects of the compounds PB01 and PB11 on variouscancer cell strains were found to be prominent at a concentration of 50μM. Even though the sensitivity of the compounds PB01 and PB05 slightlydiffers among the different cancer cell strains, the compounds PB01 andPB05 were the most effective in inhibiting cancer cell growth in thehuman non-small cell lung cancer cell strains A549 and H460, but hadnearly zero cancer cell growth inhibitory effect in non-cancer lung cellstrains MRC-5 and MRC-9, indicating that the compounds PB01 and PB11 ofExamples 5 and 3 have a cell growth inhibitory effect selectively onlyon cancer cell strains.

EXAMPLE 8 Apoptosis Effect Assay on Non-Small Cell Lung Cancer CellStrain

Whether the cancer cell growth inhibitory effects of the compounds PB01and PB11 on the non-small cell lung cancer cell strains is associatedwith apoptosis was investigated. The cell growth inhibitory effects wereevaluated by a trypan blue assay, and cell shapes were observed using aninverted microscope. The shapes of cell nuclei were observed viaHoeschst 33342 staining. Degrees of inducing apoptosis were quantized byflow cytometry (fluorescent activated cell sorting (FACS)).

As a result, IC₅₀s of the compounds PB01 and PB11 in the human nonsmallcell lung cancer cell strains A549 and H460 were 50 μM, and the timetaken to reach the IC₅₀s was 48 hours. Both of the compounds PB01 andPB11 were found to induce cell death in lung cancer cells, indicatingthat the compounds PB01 and PB11 may induce apoptosis as a cell deathmechanism.

1) Evaluation of Apoptosis Effect of PB01 and PB11 in Non-Small CellLung Cancer Cells

After about 5×10³ cells per well of each of the human non-small celllung cancer cell strains A549 and H460 were portioned into a 96-wellplate, about 10 uM to about 50 uM of the compounds PB01 or PB11 in 200ul of culture solution were added into the wells, and incubated forabout 8 to 72 hours. Cells were collected from the incubation product,and centrifuged at about 2,000 rpm for 5 minutes. After removing asupernatant, the remaining cells were mixed with 1 mL ofphosphate-buffered saline (PBS) to obtain a cell suspension, which wasthen mixed with an equal amount of 0.5% trypan blue (Gibco BRL),followed by treatment for about 1 minute. The number of live cells wascounted using a phase-contrast) microscope. The live cell counting wasrepeated three times, and an average live cell count and a standarderror were calculated using a Microsoft Excel program. Cell viabilitygraphs with respect to compound concentration and incubation time wereobtained. The same experiment was performed with 50 μM of the compoundPB01, PB11 50 μM of the compound PB11, or 150 μM of the compound PB12150 μM, and cell viability graphs with respect to compound concentrationand incubation time were obtained.

FIG. 3A illustrates graphs of cell viability with respect to testcompound concentration and incubation time in the non-small cell lungcancer cell strains A549 and H460 after incubation together with thecompounds PB01 or PB 11 of Examples 5 and 3, obtained by microscopy withtrypan blue staining.

FIG. 3B illustrates graphs of cell viability with respect to testcompound concentration and incubation time in the non-small cell lungcancer cell strain H460 after incubation together with the testcompounds PB01, PB 11, or PB12 of Examples 5, 3, and 4, obtained bymicroscopy with trypan blue staining.

Referring to FIGS. 3A and 3B, the compounds PB01, PB 11, and PB12 ofExamples 5, 3, and 4 were found to have improved cancer cell apoptosiseffects with increases in concentration and incubation time. Inparticular, remarkable cancer cell apoptosis effects were obtained inthe groups treated with 50 μM of the compound PB01 or the compound PB11.

After the incubation along with 50 μM of the compounds PB01 or PB11 for48 hours, morphological changes in the non-small cell lung cancer cellstrain H460 were observed using an inverted microscope at amagnification of 200×. The results are shown in FIG. 4.

Referring to FIG. 4, when treated with the compounds PB01 or PB11 ofExamples 5 and 3, the cancer cells appear to lose adhesion with reduceddensity, and float on the surface of the medium. This morphologicalchange seems to coincide with the cell growth inhibitory effects of thecompounds PB01 and PB11.

2) Observation on Nuclear Agglutination and Segmentation in Non-SmallCell Lung Cancer Cells by PB01 and PB11

To observe typical nuclear morphological changes from cell death, thecells treated with the compounds PB01 or PB11 of Examples 5 and 3 werecollected, followed by removing a supernatant. After 500 μl of a fixingsolution as a 1:9 mixture of a 37% formaldehyde solution and PBS buffersolution was added into the remaining cells, thoroughly mixed together,and left at room temperature for about 10 minutes for fixation. Afterthe fixed cells were centrifuged at about 2,000 rpm for about 5 minutes,the fixing solution was removed, and the remaining cells were floated ina PBS buffer solution. After dropping the floating cells onto a slideglass, followed by cytospinning at about 1,000 rpm for about 5 minutesto fix the floating cells onto the slide glass. The slide glass with thefixed cells was treated with 2 μg/mL of a Hoechst 33342 solution (Sigma,St. Louis, Mo., U.S.A), followed by staining at room temperature forabout 20 minutes. After termination of the staining, the stainingreagent was washed out, and nuclear morphological changes in the cancercells were observed by florescent microscopy (ECLIPSE E600; Nikon,Tokyo, Japan) at a 400× magnification. The results are shown in FIGS. 5and 6.

FIG. 5 illustrates fluorescent microscopic images of the non-small celllung cancer cell strain H460 at a 400× magnification obtained viaHoechst staining after incubation with 50 uM of the compounds PB01 orPB11 of Examples 5 and 3 for about 48 hours in order to observe nuclearmorphological changes in the cancer cell.

FIG. 6 illustrates graphs of degrees of nuclear agglutination andsegmentation, obtained based on the nuclear morphological changes in thenon-small cell lung cancer cell strain A549 or H460 that were observedby fluorescent microscopy at a 400× magnification via Hoechst stainingafter incubation with 50 uM of the compound PB01 or PB11 of Examples 5and 3 for about 48 hours.

Referring to FIGS. 5 and 6, in the cancer cells grown in a normal mediumnormally stained nuclei appeared distinct. However, in the cancer cellstreated with the compounds of PB01 or PB11 of Examples 5 and 3,increased apoptotic bodies due to nuclear agglutination were found,which is a typical feature observed in cells with apoptosis. Theseresults indicate that cell growth inhibitory effects and morphologicalchanges in the non-small cell lung cancer cells treated with thecompounds PB01 and PB11 are related with apoptosis.

3) Measurement of LDH Release from Non-Small Cell Lung Cancer CellsTreated with PB01 or PB11

After incubation of the non-small cell lung cancer cell strains A549 andH460 treated with the compound PB01 or PB11 for 8, 24, and 48 hours,only supernatants were recovered the culture products. The activity oflactate dehydrogenase (LDH) may be measured by detecting an amount ofLDH released from the cell cytosol with a Cytotoxicity Detection Kit(available from Roche Applied Science, Indianapolis, Ind.). The amountsof LDH release induced by the compound PB01 or PB11 were calculated as apercentage amount of extracellular LDH released from the cells withrespect to a total amount of LDH as a sum of the extracellular releasedLDH and the intracellular LDH remaining in the cells. The results areshown in FIG. 7.

FIG. 7 illustrates graphs of amount of LDH released from the non-smallcell lung cancer cell strains A549 and H460 treated with 50 uM of thecompound PB01 or PB11 of Examples 5 and 3 into the culture media whileincubation for 0 to 48 hours.

Referring to FIG. 7, in the groups (the non-small cell lung cancer cellsA549 and H460) treated with the compound PB01 or PB11, the amounts ofextracellular LDH released from the cells were increased with time,compared to the untreated groups, indicating that a cell depth mechanismin the groups treated with the compound PB01 or PB11 occurred throughapoptosis.

EXAMPLE 9 Investigation on Cell Death Mechanism in Non-Small Cell LungCancers

1) Measurement of Effects of PB01 and PB11 on Sub-G1 Phase Cell Number

After incubation of the non-small cell lung cancer cells A549 and H460treated with 50 μM of the compound PB01 or PB11 of Examples ?5 and 3 for48 hours, the non-small cell lung cancer cells A549 and H460 werecollected and floated with a 1× PBS (pH 7.4), followed by fixing atabout −20° C. with a 70% cold ethanol, washing twice with a 1× PBS, andthen staining with 10 μg/mL of propidium iodide (including 0.5% of PI,Tween-20, 100 μg/mL of 0.1% RNase) at room temperature for about 30minutes. The stained cells were analyzed by flow cytometry with aFACScan™ (available from Becton Dicknson). The results are shown in FIG.8.

FIG. 8A illustrates the results of flow cytometry on the non-small celllung cancer cells A549 after incubation with 50 μM of the compound ofPB01 or PB11 for 48 hours, followed by staining with propidium iodide.

FIG. 8B illustrates the results of flow cytometry on the non-small celllung cancer cells H460 after incubation with 50 μM of the compound ofPB01 or PB11 for 48 hours, followed by staining with propidium iodide.

Referring to FIG. 8, a sharp increase in the number of sub-G1 phasecells occurred in the non-small cell lung cancer cells A549 and H460treated with 50 μM of the compounds of PB01 or PB11, indicating thatcell death occurs through apoptosis.

2) Measurement of Dead Cell Count in Non-Small Cell Lung Cancer CellsTreated with PB01 or PB11

About 5×10⁵ cells per well of each of the non-small cell lung cancercell strains A549 and H460 were portioned into a 6-well plate and thenincubated with the compounds PB01 or PB11 for about 40 hours in the samemanner as described above. A cell layer was washed with PBS and thentreated with trypsin-EDTA solution to isolate cells. The isolated cellswere collected and stained with an Annexin V Flous Staining kit(available Roche Applied Science, Penzberg, Germany) in a dark conditionfor about 30 minutes. The stained cells were analyzed by flow cytometryusing a FACScan™ (available from Becton Dicknson) to count the number ofdead cells and then calculate an apoptosis ratio. The same experimentwas performed with 50 μM of PB01, 50 μM of PB11, or 150 μM of PB12 inthe same manner as described above. The graphs of cell viability withrespect to compound concentration and incubation time obtained based onthe experimental results are shown in FIGS. 9A and 9B.

FIG. 9A illustrates graphs of apoptosis ratio in the non-small cell lungcancer cell strains A549 and H460 incubated with 50 μM of the compoundsPB01 or PB11 of Examples 5 and 3 for about 40 hours, wherein theapoptosis ratios were calculated based on the results of flow cytometryusing FACScan™ (available Becton Dicknson) via staining with an AnnexinV Flous Staining kit in a dark condition for about 30 minutes.

FIG. 9B illustrates graphs of apoptosis ratio in the non-small cell lungcancer cell strain H460 incubated with the compound PB01, PB11, or PB12for about 40 hours, wherein the apoptosis ratios were calculated basedon the results of flow cytometry using FACScan™ (available BectonDicknson) via staining with an Annexin V Flous Staining kit in a darkcondition for about 30 minutes.

Referring to FIGS. 9A and 9B, the compounds PB01, PB11, and PB12 ofExamples 5, 2, and 4 were found to kill the non-small cell lung cancercells by inducing apoptosis.

3) Cell Death-Related Protein Assay of Non-Small Cell Lung Cancer Cells

After incubation of the non-small cell lung cancer cells A549 and H460treated with 50 μM of the compound PB01 or PB11 of Examples 5 and 3 forabout 48 hours, the resulting cell culture was added to a lysis buffer(1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) to lysecells. The resulting product was put on ice for about 30 minutes,followed by centrifugation to extract proteins and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 10%-15% gels.After the separated proteins were electrically transferred from theSDS-PAGE gels to nitrocellulose membranes at about 10V for about 2hours, the nitrocellulose membranes with the proteins were reacted in ablocking buffer (1× TBS, 0.1% Tween-20, 5% skim milk) at roomtemperature for about 1 hour, treated with primary antibodies, andreacted at about 4° C. overnight. After further reaction withhorseradish peroxidase (HRP)-conjugated anti-goat, anti-rabbit, andanti-mouse secondary antibodies for about 1 hour, the color wasdeveloped using an enhanced chemiluminescence (ECL)-plus system(available from Amersham Biosciences), followed by exposure to an X-rayfilm. The resulting western blot images are shown in FIG. 10.

FIG. 10 illustrates images obtained from western blotting followingSDS-PAGE on the proteins extracted from the non-small cell lung cancercell strain H460 incubated with 50 μM of the compound PB01 or PB11 ofExamples 5 and 3 for about 48 hours.

Based on the fact that cell death occurs through apoptosis, foundthrough the above-described experiments, Western blotting as describedabove was conducted to accurately identify changes in typicalintracellular apoptosis-related proteins during a cell death process,and an apoptosis mechanism. As a result, referring to FIG. 10, activityof caspase-8 as an apoptosis-initiating protein was detected. Theactivities of Bid, caspase-9, caspase-3, and PARP proteins involved inthe following stages of apoptosis were also detected. The resultsindicate that apoptosis is induced through the activation mechanism ofthese proteins.

EXAMPLE 10 Radiation Sensitivity Measurement

About 500 cells of non-small cell lung cancer cell strain H460 werefloated on each of 60-mm cell culture plates, and then incubated forabout 24 hours. After the cells were stably fixed on the bottom of thecell culture plate, the cells were treated with ciglitazone (10 μM),PB01(10 μM), or PB11 (5 μM, 1 μM), cultured for about 24 hours, and thenirradiated with y-radiation (3.2 Gy/min, Gammacell 3000; Atomic Energyof Canada, Ltd., Mississauga, ON, Canada) at doses of 2 Gy, 4 Gy, and 6Gy. After incubation for 14 days, followed staining using a 1% crystalviolet staining reagent solution (in methanol) to selectively countcolonies having a diameter of about 0.5 mm or larger. The colonyformation rate of the cells was calculated using the following equation,and the results are shown in FIG. 11.

SF (Surviving Fraction)=Number of Colonies Formed/Number of CellsSeeded×Plating Efficiency of the Control Group

FIG. 12 illustrates data obtained based on the results of colonycounting, representing an effect of the compound PB01 or PB11 ofExamples 5 and 3 as a radiation sensitizer when treated in combinationwith radiation. A dose increase rate of 1 or larger to 2 or lessindicates an effect as a radiation sensitizer. Therefore, the compoundsof PB01(10 μM) and PB11 (5 μM) used in combination with radiation at adose of 4 Gy were found to have a significant effect as radiationsensitizers.

FIG. 13 illustrates visually observed images of colony formation.

As described above, according to the one or more of the aboveembodiments of the present invention, a novel compound represented byFormula I may function as a PPAR-γ ligand, may have anticancer activityin various types of cancer cells, and may function as an anticancer drugwith minimum side effects due to having selective cytotoxicity only tocancer cells. The novel compound may also function as a radiationsensitizer in cancer treatment with radiotherapy.

It should be understood that the exemplary embodiments described thereinshould be considered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments.

While one or more embodiments of the present invention have beendescribed with reference to the figures, it will be understood by thoseof ordinary skill in the art that various changes in form and detailsmay be made therein without departing from the spirit and scope of thepresent invention as defined by the following claims.

1. A compound of Formula I, a pharmaceutically acceptable salt thereof,or a solvate thereof:

wherein, in Formula I, —R¹ is a C₁-C₃ alkoxy, ═O or —OH, and —R² is2H-1,2-oxazine-5-yl, 6H-1,2-oxazine-5-yl, 2H-1,3-oxazine-5-yl,4H-1,3-oxazine-5-yl, isoxazole-4-yl, or oxazole-4-yl, which isoptionally substituted with a C₁-C₃ alkyl, a C_(16l) -C₃ alkoxy, orhydroxy, wherein the compound of Formula I excludesN-[2-[[(3,5-dimethyl-4-isoxazolyl)methyl]thio]-6-benzothiazolyl]-4-methoxy-cyclohexanecarboxamideand4-methoxy-N-[2-[(3-pyridinylmethyl)thio]-6-benzothiazolyl]-cyclohexanecarboxamide.2. The compound, the pharmaceutically acceptable salt thereof, or thesolvate thereof of claim 1, wherein —R1 is methoxy.
 3. (canceled)
 4. Thecompound, the pharmaceutically acceptable salt thereof, or the solvatethereof of claim 1, wherein the compound of Formula 1 is selected fromthe group consisting of 4-oxo-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isooxazole-4-yl-methyl)sulfanyl-benzothiazole-6-yl]-amide;and 4-hydroxy-cyclohexanecarboxylic acid[2-(3,5-dimethyl-isoxazole-4-yl-methyl)sulfanyl-benzothiazole-6-yl]-amide.5. A pharmaceutical composition comprising the compound of Formula 1,the pharmaceutically acceptable salt thereof, or the solvate thereofaccording to claim
 1. 6.-10. (canceled)